Project Summary This proposal describes a five-year mentored program with the specific goal of preparing the principal investigator for an independent research career in cardiovascular medicine. The project aims to impart the skills and knowledge required for the applicant to achieve his long-term goal of using tissue engineering to investigate kinase signaling pathways. The immediate goals of this project are to 1) gain the experimental experience necessary develop engineered cardiac microtissues from human iPS cells 2) expand the applicant's knowledge base with the appropriate coursework in stem cell biology, biomedical engineering and faculty development 3) develop the necessary administrative skills required to be an independent researcher 4) formulate a body of work that will enable funding as an independent investigator. The applicant will have the resources to achieve these goals under the guidance of his mentor Professor Vunjak-Novakovic as well as a carefully selected advisory board that will foster the transition of the applicant from mentored to independent research. Project Description In preliminary work, human cardiac progenitor cells were used as a tool for screening the kinome for important drivers of cell survival in cardiovascular physiology. We identified the serine/threonine kinase, STK25, as a regulator of cardiac progenitor cell survival and used both overexpression and knockdown studies to show that STK25 controls the 5'-AMP-activated protein kinase (AMPK) pathway in these cells. This impacted cell proliferation and metabolism. STK25 was also able to bind to LKB1, an upstream regulator of AMPK. We then investigated this pathway in cardiomyocytes which we differentiated from inducible pluripotent stem cells (iPS) and found that deletion of STK25 using a CRISPR-Cas9 system negatively regulated the AMPK pathway. Furthermore, this was shown to be deleterious to the mechanical performance of cardiomyocytes in simple strain assays. The goal of this proposal is to investigate the mechanism behind how STK25 regulates the AMPK pathway and to demonstrate the physiologic significance of that regulation in cellular based assays. Harnessing our expertise in tissue engineering, we will generate mature, adult-like human myocardial microtissues from iPS cells and use them to model the physiological impact of this signaling pathway with genetic manipulation of both STK25 and AMPK. This data will demonstrate the innovation of CPC's as a surrogate for cardiac signaling exploration, the mechanism by which STK25 impacts cardiomyocyte function and the therapeutic potential of this pathway on cardiomyocyte performance.